Method of modulating cell survival and reagents useful for same

ABSTRACT

The present invention relates generally to a method for modulating cell survival. Modulation of cell survival includes inducing, enhancing or otherwise promoting cell survival such as the survival of neural cells as well as facilitating cell death such as the death of targeted cancer cells. The modulation of cell survival is mediated by a region identified on the p75 neurotrophin receptor (p75 NTR ) required for death signalling. The present invention further provides genetic molecules which encode the death signalling region of p75 NTR  which are useful in antagonizing death signal function as well as promoting cell death when expressed in targeted cells. The present invention also contemplates recombinant peptides, polypeptides and proteins s well as chemical equivalents, derivatives and homologues thereof which comprise the death signalling portion of p75 NTR . Particularly useful molecules of the present invention comprise peptides corresponding to soluble forms of the death signalling portion of p75 NTR . These molecules antagonize p75 NTR -mediated cell death.

FIELD OF THE INVENTION

[0001] The present invention relates generally to a method formodulating cell survival. Modulation of cell survival includes inducing,enhancing or otherwise promoting cell survival such as the survival ofneural cells as well as facilitating cell death such as the death oftargeted cancer cells. The modulation of cell survival is mediated by aregion identified on the p75 neurotrophin receptor (p75^(NTR)) requiredfor death signalling. The present invention further provides geneticmolecules which encode the death signalling region of p75^(NTR) whichare useful in antagonising death signal function as well as promotingcell death when expressed in targeted cells. The present invention alsocontemplates recombinant peptides, polypeptides and proteins as well aschemical equivalents, derivatives and homologues thereof which comprisethe death signalling portion of p75^(NTR). Particularly useful moleculesof the present invention comprise peptides corresponding to solubleforms of the death signalling portion of p75^(NTR). These moleculesantagonise p75^(NTR)-mediated cell death.

BACKGROUND OF THE INVENTION

[0002] Bibliographic details of the publications-numerically referred toin this specification are collected at the end of the description.

[0003] The subject specification contains nucleotide and amino acidsequence information prepared using the programme Patentln Version 2.0,presented herein after the bibliography. Each nucleotide or amino acidsequence is identified in the sequence listing by the numeric indicator<210> followed by the sequence identifier (e.g. <210>1, <210>2, etc).The length, type of sequence (DNA, protein (PRT), etc) and sourceorganism for each nucleotide or amino acid sequence are indicated byinformation provided in the numeric indicator fields <211>, <212> and<213>, respectively. Nucleotide and amino acid sequences referred to inthe specification are defined by the information provided in numericindicator field <400> followed by the sequence identifier (eg. <400>1,<400>2, etc).

[0004] The increasing sophistication of recombinant DNA technology isgreatly facilitating research and development in the medical and alliedhealth fields. This is particularly the case in the development ofrecombinant cytokines and growth factors for use in the treatment ofdiabetes, acquired immunodeficency syndrome (AIDS) and a number ofcancers.

[0005] However, despite this developing knowledge of cytokine and growthfactor effector molecules, their full exploitation requires anunderstanding of the corresponding cellular receptors and the complexbiochemical and physiological signalling pathways initiated followinginteraction with ligands or following other stimulation such as disease,receptor aggregation or trauma.

[0006] A number of soluble trophic factors have been shown to exhibit aneffect on neural survival in vivo. Many of these factors act directly onthe developing neuron within, for example, the dorsal root ganglia(DRG). One factor of particular importance is nerve growth factor (NGF)[1]. The p75 neurotrophin receptor (hereinafter referred to as“p75^(NTR)”), which is capable of associating with trk growth factorreceptors, facilitates high affinity NGF binding and survivalsignalling. Although NGF has been proposed as a potential therapeuticmolecule to promote survival of neurons, NGF is a multifunctionalmolecule and its pleiotrophy may adversely effect a range of non-neuralcells.

[0007] p75^(NTR) is also multifunctional. It has now been shown thatp75^(NTR) is capable of acting as a death receptor. Elevated p75^(NTR)expression results in increased cell death in vitro and in vivo [2-4].Furthermore, down-regulation of p75^(NTR) prevents neural death aftergrowth-factor withdrawal or axotomy [5, 6]. Consistent with the dualfunctions of p75^(NTR), mice with deleted p75^(NTR) genes have adramatic reduction of NGF dependent neurons, such as dorsal rootganglia, but increased numbers of other neuron populations (sympatheticand basal forebrain neurons) suggesting lack of naturally occurring celldeath [7, 8]. p75^(NTR) is also implicated in mediating death of neural,oligodendrocytes and Schwann cells [8, 9].

[0008] p75^(NTR) is a member of the tumor necrosis factor (TNF)receptor/Fas superfamily, showing homology not only to the extracellularligand binding domain but also to a cytoplasmic motif known as the“death domain”, so termed because of the cytotoxic actions of proteinscontaining the domain [9].

[0009] There is an accumulating body of evidence which suggests thatp75^(NTR) is involved in mediating cell death in a variety ofdegenerative diseases. During adulthood, p75^(NTR) expression isdown-regulated in most brain areas but is rapidly induced in ischemia(stroke) and results in transient increased p75^(NTR) expression andapoptosis, as do both peripheral and motor nerve lesions [10-12].p75^(NTR) is also up regulated in patients with MND [13], and inexperimental allergic encephalomyelitis (a model of multiple sclerosis;[14]). Intriguingly, in the basal forebrain and hippocampus, areasinvolved in learning and memory, p75^(NTR) is highly expressed in agedrodents and in Alzheimer's patients, where extensive neural death isoccurring [15, 16]. These data suggest that p75^(NTR) is involved notonly in normal developmental cell death, but may mediate the cell deathoccurring after injury or in neurodegenerative disease.

[0010] In work leading up to the present invention, the inventors soughtto elucidate the region on p75NTR which mediates death signalling. Theinventors surprisingly determined that the death signal is not thecytoplasmic motif known as the death domain [9] but is a region adjacentthe membrane domain on p75^(NTR) The identification of this regionprovides for an opportunity to modulate cell survival by antagonisingthe death signalling region or promoting apoptosis by providing cellswith the genetic material to express the death signalling regionadjacent, proximal or otherwise juxtaposed or associated with themembrane or to express the death signalling region in multimeric form.

SUMMARY OF THE INVENTION

[0011] Throughout this specification, unless the context requiresotherwise, the word “comprise”, or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedelement or integer or group of elements or integers but not theexclusion of any other element or integer or group of elements orintegers.

[0012] One aspect of the present invention provides an isolated nucleicacid molecule comprising a sequence of nucleotides or complementarysequence of nucleotides which encodes an amino acid sequence which iscapable of signalling, inducing or otherwise facilitating the death of acell in which said amino acid sequence is adjacent, proximal orotherwise juxtaposed to the membrane of said cell or when said aminoacid sequence is in multimeric form.

[0013] Another aspect of the present invention is directed to a nucleicacid molecule comprising a sequence of nucleotides or complementarysequence of nucleotides which encodes a peptide, polypeptide or proteincapable of signalling, inducing or otherwise facilitating death of acell in which it is expressed wherein said peptide, polypeptide orprotein comprises a membrane associating portion and/or amultimer-forming portion and a portion which corresponds to all or partof the cytoplasmic region of p75^(NTR) or a functional equivalent,derivative or homologue thereof.

[0014] Yet another aspect of the present invention contemplateshomologues, analogues and derivatives of a nucleic acid molecule whichencodes a peptide, polypeptide or protein which is capable of signallinginducing or otherwise facilitating death of a cell in which it isexpressed wherein said peptide, polypeptide or protein comprises amembrane associating portion and/or a multimer-forming portion and aportion which corresponds to all or part of the cytoplasmic region ofp75^(NTR) or a functional equivalent, derivative or homologue thereof.

[0015] A further aspect of the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides which encodesan amino acid sequence which inhibits or reduces p75^(NTR)-mediated celldeath wherein said amino acid sequence is a soluble form of thep75^(NTR) receptor corresponding to an intracellular region adjacent,proximal or otherwise juxtaposed to the membrane of said cell.

[0016] Still another aspect of the present invention provides a nucleicacid molecule comprising a nucleotide sequence or complementarynucleotide sequence which is substantially as set forth in <400>3 or isa nucleotide sequence capable of hybridising to <400>3 or itscomplementary form under low stringency conditions or is a nucleotidesequence having at least 60% identity to <400>3.

[0017] Still yet another aspect of the present invention contemplates anucleic acid molecule comprising a nucleotide sequence or acomplementary form thereof, which nucleotide sequence encodes an aminoacid sequence substantially as set forth in <400>4 or a derivative,homologue or chemical equivalent thereof or an amino acid sequencehaving at least 60% identity thereto.

[0018] Even yet another aspect of the present-invention provides agenetic construct comprising an isolated nucleic acid molecule whichcomprises a sequence of nucleotides which corresponds or iscomplementary to a death signal region from p75^(NTR) or a homologue,analogue or derivative thereof.

[0019] Another aspect of the present invention contemplates an isolatedpeptide, polypeptide or protein comprising the cytoplasmic region ofp75^(NTR) which signals, induces or otherwise facilitates cell deathwhen said peptide, polypeptide or protein is adjacent, proximal orotherwise juxtaposed to a membrane-associating region such as fromp75^(NTR) or other membrane molecule and/or said peptide, polypeptide orprotein is capable of forming multimers or a derivative, homologue,chemical equivalent or analogue of said peptide, polypeptide or protein.This aspect of the present invention does not extend to the full lengthp75^(NTR).

[0020] Still another aspect of the present invention contemplates amethod for inhibiting, reducing or otherwise antagonising ap75^(NTR)-mediated death signal in a neural cell, said method comprisingintroducing a nucleic acid molecule capable of being expressed to anexpression product which corresponds to a non-membrane associated formof the p75^(NTR) death signal region or a derivative, functionalequivalent or homologue thereof.

[0021] Yet another aspect of the invention contemplates a method forinhibiting, reducing or otherwise antagonising a p75^(NTR)-mediateddeath signal in a neural cell, said method comprising contacting a cellcarrying a p75^(NTR) with a death signal-inhibiting effective amount ofa molecule capable of antagonising the death signal of p75^(NTR) or acomponent of the death signalling pathway.

[0022] Even still another aspect of the present invention provides abiological composition comprising a genetic molecule capable ofexpressing a p75^(NTR) death signal antagonist or a p75^(NTR) deathsignal.

[0023] Another aspect of the present invention is directed to abiological composition comprising a molecule capable of antagonisingp75^(NTR)-mediated death signalling of a cell.

[0024] Yet still another aspect of the present invention contemplates amethod for modulating p75^(NTR)-mediated death signal in a neural cell,said method comprising administering an agent which antagonises oragonises cleavage of the extracellular domain of p₇₅ ^(NTR).

[0025] Still another aspect of the present invention provides a methodfor inhibiting, reducing or otherwise antagonising p75^(NTR)-mediateddeath signal in a neural cell, said method comprising administering apeptide, polypeptide or protein or analogues or mimetics thereof whichcorrespond to a non-membrane associated form of the p75^(NTR) deathsignal region or a derivative, functional equivalent or homologuethereof.

[0026] Another aspect of the present invention provides peptideantagonists of the p75^(NTR) death signal or functional analogues ormimetics thereof.

[0027] The terms “c35” and “35 mer” are used interchangeably herein torefer to 35 amino acid domain juxtaposed to the membrane. When insoluble form, this peptide is referred to as soluble c35 or 35 mer. Thenucleotide and amino acid sequence of c35 are shown in <400>7 and<400>8, respectively. The term “29 mer” refers to a truncated form ofthe 35 mer. Six amino acids have been deleted from the C-terminal end.The nucleotide and amino acid sequence of 29 mer are shown in <400>11and <400>12, respectively. The present invention extends to isolatedforms of c35 and the 29 mer, to compositions comprising same and togenetic sequences encoding same.

BRIEF DESCRIPTION OF THE FIGURES

[0028]FIG. 1 is a diagrammatic representation showing plasmid constructswith and without the death signalling region. The black region is theputative “death domain” [9] but which is not directly involved inp75^(NTR) mediated cell death.

[0029]FIG. 2 is a graphical representation showing survival of DRGneurons 17 hours after microinfection and cultured in LIF. The data showthat the amino acid domain juxtaposed to the membrane is required fordeath signalling rather than the putative “death domain” [9].

[0030]FIG. 3 is a graphical representation showing DRG survival 16 hoursafter microinjection and cultured in LIF. The data show that over 90% ofcells die when expressing the death signal linked to the membrane.

[0031]FIG. 4 is a graphical representation showing DRG survival 20 hoursafter microinjection and cultured in LIF. These data show that when thedeath signal is not associated with the membrane, that the ability toinduce death is removed.

[0032]FIG. 5 is a graphical representation showing that the c35 solubleprotein (i.e. p75^(NTR) death signal region) inhibits death signallingmediated by p75^(NTR).

[0033]FIG. 6 is a graphical representation showing that soluble c35inhibits p75^(NTR)-mediated death signalling.

[0034]FIG. 7 is a graphical representation showing protection ofmembrane-bound killing-domain by a soluble 35 amino acid peptide and asoluble 29 amino acid peptide. The cells were subjected tomicroinjection of sptc35 or GFP followed 30 minutes later by eitherpeptide c35 or the 29 mer peptide.

[0035]FIG. 8 is a graphical representation showing that peptide 29 whichhas a palmitoyl group at the membrane (amino) end and which facilitatesassociation with the membrane mediates to cell death. In contrast, thesoluble 35 amino acid molecule tends to protect the cells. F, Fluorotagged; pen, penetratin.

[0036]FIG. 9 is a graphical representation showing the palmitoylated 29mer fused to penetratin mediates specific killing whereasnon-palmitoylated 29 mer blocks cell death. Cells were treated with 2 μMpeptide for 1-2 hours then washed. pen, penetratin, F29, 29 mer, Palm,palmitoylation.

pen F29

pen F29 Palm

pen gp130 Palm

pen

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0037] The present invention arose in part following an investigation ofthe neurotrophin receptor, p75^(NTR), in its capacity as a deathsignalling protein. Although the p75^(NTR) molecule comprises a putativedeath domain [9], in accordance with the present invention, this deathdomain is not directly associated with p75^(NTR)-mediated cell death.Rather, a region adjacent, proximal or otherwise juxtaposed to themembrane domain of p75^(NTR) is required for cell death. The nucleotideand corresponding amino acid sequence of the death domain [9] is shownin <400>9 and <400>10, respectively.

[0038] Accordingly, one aspect of the present invention provides anisolated nucleic acid molecule comprising a sequence of nucleotides orcomplementary sequence of nucleotides which encode an amino acidsequence which is capable of signalling, inducing or otherwisefacilitating the death of a cell in which said amino acid sequence isadjacent, proximal or otherwise juxtaposed to the membrane of said cellor when said amino acid sequence is in multimeric form.

[0039] Reference herein to the signalling, inducing or otherwisefacilitating the death of a cell or a death signal is meant to beconstrued in its broadest sense meaning that the amino acid sequenceplays a role in a pathway leading to cell death. The death signal mayalso be regarded as an apoptopic signal. Although not wishing to limitthe present invention to any one theory or mode of action, it isproposed herein that there is a pathway from p75^(NTR) activation tocaspase activation and cellular degeneration. p75^(NTR)-mediated celldeath may also occur directly or indirectly via Bcl-2.

[0040] The present specification refers interchangeably to death signal,death signal region, signalling, inducing or otherwise facilitating thedeath of a cell and c35.

[0041] The nucleic acid molecule of the present invention may encode anon-full length p75^(NTR) molecule although to facilitate cell death,the nucleic acid molecule must encode all or part of the cytoplasmicportion of the p75^(NTR) molecule and a sufficient amount of themembrane domain such that the region referred to herein as the deathsignal is membrane associated. A “part” of the cytoplasmic domain ofp75^(NTR) includes all or a death-inducing functional part of a 35 aminoacid region juxtaposed to the membrane domain. An example of a part ofthe 35 amino acid region is a truncated form. One such form is referredto herein as the “29 mer”. Alternatively, the cytoplasmic domain of thep75^(NTR) molecule is in multimeric form or capable of formingmultimers. A multimer comprises two or more copies of the molecule suchas a dimer, trimer or larger copy molecule.

[0042] The term “membrane associated” means that the death signal isadjacent, proximal or otherwise juxtaposed to the membrane of a cellexpressing the nucleic acid molecule.

[0043] The “death signal region” and other related terms are used hereinto describe functionally the region of the cytoplasmic portion ofp75^(NTR) which is adjacent, proximal or otherwise juxtaposed to aregion of p75^(NTR) which associates with the membrane or whichcytoplasmic portion is in multimeric form. The death signal region isnot the same portion of the molecules as the “death domain” [9] althoughthere may be functional similarities in death signalling.

[0044] Accordingly, another aspect of the present invention is directedto a nucleic acid molecule comprising a sequence of nucleotides orcomplementary sequence of nucleotides which encodes a peptide,polypeptide or protein capable of signalling, inducing or otherwisefacilitating death of a cell in which it is expressed wherein saidpeptide, polypeptide or protein comprises a membrane associating portionand/or a multimer-forming portion and a portion which corresponds to allor part of the cytoplasmic region of p75^(NTR) or a functionalequivalent, derivative or homologue thereof.

[0045] In order to signal, induce or otherwise facilitate death of acell, the death signal region is preferably adjacent, proximal orotherwise juxtaposed to the cell membrane. This may be facilitated bymodifying a peptide such that it associates with the membrane. Oneexample of this type of modification is palmi toylation. This puts apalmitoyl group at the membrane (amino) end of the peptide.

[0046] Accordingly, another aspect of the present invention contemplatesplamitoylated peptides, polypeptides or proteins comprising all or partof the death signal region of p75^(NTR). Such peptides are particularlyuseful in promoting cell death.

[0047] The present invention also extends to multimeric forms of deathsignal peptides, polypeptides and proteins and attachments whichfacilitate same. A multimer comprises two or more molecules. The presentinvention also extends to cleavage forms of the full length p75^(NTR)molecule.

[0048] In one embodiment, the membrane portion is derived from p75^(NTR)or a functional equivalent, derivative or homologue thereof. In anotherembodiment, the membrane domain is from another molecule such as areceptor or other ligand-binding molecule. Examples of receptorsaccording to this aspect of the present invention include cytokinereceptors (e.g. the Leukaemia Inhibitory Factor (LIF) receptor,interleukin receptor, and colony-stimulating factor receptors). Examplesof ligand-binding molecules include immunoglobulins and T cellreceptors.

[0049] When in multimeric form, the molecule-is only optionallyassociated with the membrane to effect cell death.

[0050] The nucleic acid molecule may comprise cDNA or genomic DNA or maycomprise ribonucleotides such as mRNA. The nucleic acid molecule may bederived from a cDNA or genomic molecule encoding p75^(NTR) or aderivative or homologue thereof or may be prepared by the stepwiseaddition of nucleotides in a defined sequence.

[0051] The nucleic acid molecule of the present invention may also beconsidered as corresponding to a “gene”.

[0052] Reference herein to a “gene” is to be taken in its broadestcontext and includes:

[0053] (i) a classical genomic gene consisting of transcriptional and/ortranslational regulatory sequences and/or a coding region and/ornon-translated sequences (i.e. introns, 5′- and 3′-untranslatedsequences);

[0054] (ii) mRNA or cDNA corresponding to the coding regions (i.e.exons) optionally comprising 5′- or 3′-untranslated sequences of thegene; or

[0055] (iii) an amplified DNA fragment or other recombinant nucleic acidmolecule produced in vitro and comprising all or a part of the codingregion and/or 5′- or 3′-untranslated sequences of the gene.

[0056] The term “gene” is also used to describe synthetic or fusionmolecules encoding all or part of a functional product. A functionalproduct is one which comprises a sequence of nucleotides or iscomplementary to a sequence of nucleotides which encodes a functionaldeath signal from p75^(NTR) or its derivative or homologue.

[0057] The nucleotide sequence of the present invention may correspondto the cDNA or genomic sequence of a gene encoding p75^(NTR) or a deathsignal region thereof or may be subjected to mutagenesis to producesingle or multiple nucleotide substitutions, deletions and/or additions.Nucleotide insertional derivatives of the nucleic acid molecule of thepresent invention include 5′ and 3′ terminal fusions as well asintra-sequence insertions of single or multiple nucleotides. Insertionalnucleotide sequence variants are those in which one or more nucleotidesare introduced into a predetermined site in the nucleotide sequencealthough random insertion is also possible with suitable screening ofthe resulting product. Deletional variants are characterised by theremoval of one or more nucleotides from the sequence. Substitutionalnucleotide variants are those in which at least one nucleotide in thesequence has been removed and a different nucleotide inserted in itsplace. Such a substitution may be “silent” in that the substitution doesnot change the amino acid defined by the codon. Alternatively,substituents are designed to alter one amino acid for another similaracting amino acid, or amino acid of like charge, polarity, orhydrophobicity.

[0058] Accordingly, another aspect of the present invention contemplateshomologues, analogues and derivatives of a nucleic acid molecule whichencodes a peptide, polypeptide or protein which is capable ofsignalling, inducing or otherwise facilitating death of a cell in whichit is expressed wherein said peptide, polypeptide or protein comprises amembrane associating portion and/or multimer-forming portion and aportion which corresponds to all or part of the cytoplasmic region ofp75^(NTR) or a functional equivalent, derivative or homologue thereof.

[0059] For the present purpose, “homologues” of a nucleic acid moleculeas herein defined or of a nucleotide sequence shall be taken to refer toan isolated nucleic acid molecule which is substantially the same as thenucleic acid molecule of the present invention or its complementarynucleotide sequence, notwithstanding the occurrence within saidsequence, of one or more nucleotide substitutions, insertions,deletions, or rearrangements.

[0060] “Analogues” of a nucleic acid molecule as herein defined or of anucleotide sequence set forth herein shall be taken to refer to anisolated nucleic acid molecule which is substantially the same as anucleic acid molecule of the present invention or its complementarynucleotide sequence, notwithstanding the occurrence of anynon-nucleotide constituents not normally present in said isolatednucleic acid molecule, for example carbohydrates, radiochemicalsincluding radionucleotides, reporter molecules such as, but not limitedto DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

[0061] “Derivatives” of a nucleic acid molecule as herein defined or ofa nucleotide sequence set forth herein shall be taken to refer to anyisolated nucleic acid molecule which contains significant sequencesimilarity to said sequence or a part thereof. Generally, the nucleotidesequence of the present invention may be subjected to mutagenesis toproduce single or multiple nucleotide substitutions, deletions and/orinsertions. Nucleotide insertional derivatives of the nucleotidesequence of the present invention include 5′ and 3′ terminal fusions aswell as intra-sequence insertions of single or multiple nucleotides ornucleotide analogues. Insertional nucleotide sequence variants are thosein which one or more nucleotides or nucleotide analogues are introducedinto a predetermined site in the nucleotide sequence of said sequence,although random insertion is also possible with suitable screening ofthe resulting product being performed. Deletional variants arecharacterised by the removal of one or more nucleotides from thenucleotide sequence. Substitutional nucleotide variants are those inwhich at least one nucleotide in the sequence has been removed and adifferent nucleotide or nucleotide analogue inserted in its place.

[0062] In one embodiment, the derivatives encode a peptide, polypeptideor protein which induces cell death. In another embodiment, thederivatives do not induce cell death but antagonise the death signal.

[0063] According to this latter embodiment, there is provided anisolated nucleic acid molecule comprising a sequence of nucleotideswhich encodes an amino acid sequence which inhibits or reducesp75^(NTR)-mediated cell death wherein said amino acid sequence is asoluble form of the p75^(NTR) receptor corresponding to an intracellularregion adjacent, proximal or otherwise juxtaposed to the membrane ofsaid cell.

[0064] The nucleic acid molecule of the present invention may be basedon a nucleotide sequence of the gene or cDNA encoding p75^(NTR) from anyanimal such as from mammals. Preferred mammals include humans, primates,livestock animals (e.g. cows, sheep, horses, pigs, donkeys, goats),laboratory test animals (e.g. rabbits, mice, rats, guinea pigs,hamsters), companion animals (e.g. dogs, cats) and captive wild animals.

[0065] A particularly preferred sequence is from human or primate ormurine p75^(NTR).

[0066] Although not wishing to limit the present invention to any onetheory or mode of action, it is proposed that the extracellular domainof p75^(NTR) may be cleaved off resulting in active death signal (seeZupan et al [20]). Accordingly, by antagonising cleavage, cell death maybe prevented or at least delayed or inhibited. Conversely, for targetedcancer cells, an agonist of p75^(NTR) extracellular domain cleavagewould promote cell death.

[0067] Accordingly, another aspect of the present invention contemplatesa method for modulating p75^(NTR)-mediated death signal in a neuralcell, said method comprising administering an agent which antagonises oragonises cleavage of the extracellular domain of p75^(NTR).

[0068] Preferably, to prevent neural cell death, extracellular p75^(NTR)cleavage is antagonised.

[0069] The present invention is exemplified using a nucleotide sequencefrom rat p75^(NTR) cDNA. This is done, however, with the understandingthat the nucleotide sequence may be from p75^(NTR) genomic or cDNA fromany animal.

[0070] Accordingly, another aspect of the present invention provides anucleic acid molecule comprising a nucleotide sequence or acomplementary form thereof wherein said nucleotide sequence is capableof hybridising to <400>1 or a complementary form thereof under lowstringency conditions, such as at 42° C.

[0071] The nucleotide sequence set forth in <400>1 is the cDNA sequenceencoding p75^(NTR). The nucleic acid molecule according to this aspectof the present invention does not extend to the full length p75^(NTR)cDNA sequence but comprises a portion which encodes an amino acidsequence which signals, induces or otherwise facilitates cell death whenassociated with a membrane portion of p₇₅ ^(NTR) or other molecules.

[0072] Accordingly, another aspect of the present invention provides anucleic acid molecule comprising a nucleotide sequence or complementarynucleotide sequence which is substantially as set forth in <400>7 or isa nucleotide sequence capable of hybridising to <400>7 or acomplementary form thereof under low stringency conditions such as at42° C. or is a nucleotide sequence having at least 60% identity to<400>7.

[0073] The nucleotide sequence set forth in <400>7 is the death signaldefined herein associated with p75^(NTR). This sequence encodes a 35amino acid region also referred to herein as “c35”. Truncated forms ofc35 are also contemplated by the present invention such as a 25-30 aminoacid molecules. One particular example is a 29 mer which lacks carboxyterminal amino acids 30 to 35. As stated above, the present inventionextends to palmitoylated c35 and its derivatives as well as moleculesfused with molecules to facilitate membrane passage such as penetratinand the TAT protein from HIV.

[0074] Reference herein to a low stringency such as at 42° C. includesand encompasses from at least about 0% v/v to at least about 15% v/vformamide and from at least about 1M to at least about 2M salt forhybridisation, and at least about 1M to at least about 2M salt forwashing conditions. Alternative stringency conditions may be appliedwhere necessary, such as medium stringency, which includes andencompasses from at least about 16%-v/v to at least about 30% v/vformamide and from at least about 0.5M to at least about 0.9M salt forhybridisation, and at least about 0.5M to at least about 0.9M salt forwashing conditions, or high stringency, which includes and encompassesfrom at least about 31% v/v to at least about 50% v/v formamide and fromat least about 0.01M to at least about 0.15M salt for hybridisation, andat least about 0.01M to at least about 0.15M salt for washingconditions. Preferably, low stringency is determined at 42° C.

[0075] The present invention further contemplates a nucleic acidmolecule comprising a nucleotide sequence or a complementary formthereof, which nucleotide sequence encodes an amino acid sequencesubstantially as set forth in <400>8 or a derivative, homologue orchemical equivalent thereof or an amino acid sequence having at least60% identity thereto.

[0076] The amino acid sequence of <400>8 corresponds to the amino acidsequence of the p75^(NTR) death signal.

[0077] The term “identity” as used herein includes exact identitybetween compared sequences at the nucleotide or amino acid level. Wherethere is non-identity at the nucleotide level, the term “similarity” mayalso be used and includes differences between sequences which result indifferent amino acids that are nevertheless related to each other at thestructural, functional, biochemical and/or conformational levels. Wherethere is non-identity at the amino acid level, “similarity” includesamino acids that are nevertheless related to each other at thestructural, functional, biochemical and/or conformational levels. In aparticularly preferred embodiment, nucleotide and sequence comparisonsare made at the level of identity rather than similarity. Any number ofprograms are available to compare nucleotide and amino acid sequences.Preferred programs have regard to an appropriate alignment. One suchprogram is Gap which considers all possible alignment and gap positionsand creates an alignment with the largest number of matched bases andthe fewest gaps. Gap uses the alignment method of Needleman and Wunsch[17]. Gap reads a scoring matrix that contains values for every possibleGCG symbol match. GAP is available on ANGIS (Australian National GenomicInformation Service) at website http://mel1.angis.org.au.

[0078] The present invention further comprises a nucleic acid moleculecomprising the nucleotide sequence:

{n₁ - - - n_(x)}_(b)a{n′_(l) - - - n′_(y)}_(c)a{n″_(l) - - - n″_(z)}_(d)

[0079] wherein

[0080] {n₁ - - - n_(x)} is a sequence of x nucleotides encoding anextracellular portion of a receptor or ligand-binding molecule;

[0081] {n′_(l) - - - n′_(y)} is a sequence of y nucleotides encoding atransmembrane peptide, polypeptide or protein or a molecule capable ofinducing multimerisation;

[0082] {n″_(l) - - - n″_(z)} is a sequence of z nucleotides comprising anucleotide sequence substantially as set forth in <400>7 or a nucleotidesequence encoding an amino acid sequence substantially as set forth in<400>8 or a nucleotide sequence capable of hybridising to <400>7 or acomplementary form thereof under low stringency conditions such as at42° C. or a nucleotide sequence having at least 60% identity to <400>7;

[0083] b, c and d may be the same or difference and each is 0, 1 or >1;

[0084] x, y and z may be the same or different and each is 0, 1 or >1;

[0085] a is a nucleotide bond;

[0086] wherein when c is 1 or >1 and d is 1 or >1 and wherein when themolecule is expressed in a neural cell, the expression product signals,induces or otherwise facilitates cell death.

[0087] Preferably, {n₁ - - - n_(x)} comprises the nucleotide sequencesubstantially as set forth in <400>3 or is a nucleotide sequence havingat least about 60% identity thereto or is capable of hybridising to<400>3 or its complementary form under low stringency conditions such asat 42° C.

[0088] Preferably, {n′_(l) - - - n′_(y)} comprises the nucleotidesequence substantially as set forth in <400>5 or is a nucleotidesequence having at least about 60% identity thereto or is capable ofhybridising to <400>5 or its complementary form under low stringencyconditions such as at 42° C.

[0089] The nucleotide sequences {n₁ - - - n_(x)}, {n′_(l) - - - n′_(y)}and {n″₁ - - - n″_(z)} may be in any order and in any combination.

[0090] For the production of a recombinant peptide, polypeptide orprotein comprising the death signal, the nucleic acid molecule of thepresent invention is placed, in the sense orientation, in operableconnection with a suitable promoter sequence and introduced into asuitable expression system, for example a bacterial, yeast, baculovirus,plant, animal or other expression system.

[0091] Accordingly, a further aspect of the present invention provides agenetic construct comprising an isolated nucleic acid molecule whichcomprises a sequence of nucleotides which corresponds or iscomplementary to a death signal region from p75^(NTR) or a homologue,analogue or derivative thereof.

[0092] According to this embodiment, the coding region of the deathsignal from p75^(NTR) may be placed in operable connection with apromoter sequence such that a gene product is capable of being expressedunder the control of said promoter sequence.

[0093] Optionally, said genetic construct further comprises a terminatorsequence.

[0094] In the present context, the term “in operable connection with” isused to indicate that expression of the isolated nucleotide sequence isunder the control of the promoter sequence with which it is connected.

[0095] The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active inplant cells are known and described in the literature. They may beisolated from bacteria, fungi, viruses, animals and/or plants.

[0096] Examples of terminators particularly suitable for use in thegenetic constructs of the present invention include the SV40polyadenylation signal, amongst others.

[0097] Reference herein to a “promoter” is to be taken in its broadestcontext and includes the transcriptional regulatory sequences of aclassical genomic gene, including the TATA box which is required foraccurate transcription initiation in eukaryotic cells, with or without aCCAAT box sequence and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers). For expression inprokaryotic cells, such as bacteria, the promoter should at leastcontain the −35 box and −10 box sequences.

[0098] A promoter is usually, but not necessarily, positioned upstreamor 5′, of the nucleotide sequence encoding the death signal ofp75^(NTR), the expression of which it regulates. Furthermore, theregulatory elements comprising a promoter are usually positioned within2 kb of the start site of transcription of the gene.

[0099] In the present context, the term “promoter” is also used todescribe a synthetic or fusion molecule, or derivative which confers,activates or enhances expression of an isolated nucleic acid molecule,in a cell, such as a plant, animal, insect, fungal, yeast or bacterialcell. Preferred promoters may contain additional copies of one or morespecific regulatory elements, to further enhance expression of a nucleicacid molecule which expression it regulates and/or to alter the spatialexpression and/or temporal expression of same. For example, regulatoryelements which confer copper inducibility may be placed adjacent to aheterologous promoter sequence driving expression of a nucleic acidmolecule, thereby conferring copper inducibility on the expression ofsaid molecule.

[0100] Placing an isolated nucleic acid molecule under the regulatorycontrol of a promoter sequence means positioning said molecule such thatexpression is controlled by the promoter sequence. Promoters aregenerally positioned 5′ (upstream) to the genes that they control. Inthe construction of heterologous promoter/structural gene combinationsit is generally preferred to position the promoter at a distance fromthe gene transcription start site that is approximately the same as thedistance between that promoter and the gene it controls in its naturalsetting, i.e., the gene from which the promoter is derived. As is knownin the art, some variation in this distance can be accommodated withoutloss of promoter function. Similarly, the preferred positioning of aregulatory sequence element with respect to a heterologous gene to beplaced under its control is defined by the positioning of the element inits natural setting, i.e., the genes from which it is derived. Again, asis known in the art, some variation in this distance can also occur.

[0101] Examples of promoters suitable for use in genetic constructs ofthe present invention include viral, fungal, bacterial, animal and plantderived promoters capable of functioning in plant, animal, insect,fungal, yeast or bacterial cells. The promoter may regulate theexpression of the nucleic acid molecule constitutively, ordifferentially with respect to the tissue in which expression occurs or,with respect to the developmental stage at which expression occurs, orin response to external stimuli such as physiological stresses, or plantpathogens, or metal ions, amongst others.

[0102] Preferably, the promoter is capable of regulating expression of anucleic acid molecule in a yeast or bacterial cell.

[0103] Examples of preferred promoters include the bacteriophage T7promoter, bacteriophage T3 promoter, SP6 promoter, lac promoter, tacpromoter, SV40 early promoter, and the like.

[0104] The genetic construct contemplated herein is introduced into asuitable expression system for a time and under conditions sufficientfor expression of said death signal or inhibitor portion from p75^(NTR)to occur.

[0105] The genetic construct may also comprise a nucleotide sequencecorresponding to all or part of the membrane domain of p75^(NTR) orother membrane molecules.

[0106] Accordingly, a further aspect of the invention contemplates arecombinant peptide, polypeptide or protein produced by expressing theisolated nucleic acid molecule herein described in a suitable host cell.The present invention extends also to a synthetic peptide fragment ofsaid recombinant gene product.

[0107] The present invention further contemplates an isolated peptide,polypeptide or protein comprising the cytoplasmic region of p75^(NTR)which signals, induces or otherwise facilitates cell death when saidpeptide, polypeptide or protein is adjacent, proximal or otherwisejuxtaposed to a membrane associating region such as from p75^(NTR) orother membrane molecule and/or is in multimeric form or a derivative,homologue, chemical equivalent or analogue of said peptide, polypeptideor protein. This aspect of the present invention does not extend to thefull length p75^(NTR).

[0108] Suitable molecules according to this aspect of the presentinvention include a peptide, polypeptide or protein corresponding to asoluble form of the death signalling region of p75^(NTR) or a moleculecapable of antagonising that region or a component of the deathsignalling pathway. An example of a possible component of the deathsignalling pathway is Bcl-2.

[0109] The peptide, polypeptide or protein of this aspect of the presentinvention is useful inter alia as a therapeutic molecule to antagonisep₇₅ ^(NTR)-mediated death signalling. For example, the peptide,polypeptide or protein may themselves be administered to directlyantagonise p75^(NTR)-mediated death signalling or the peptide,polypeptide or protein may need to be chemically modified to facilitatepenetration into the cell. One such chemical modification is fusion toor co-expression with penetratin or the TAT protein from HIV.Alternatively, the death signalling region of p75^(NTR) may be used toscreen for antagonists of this region. Such antagonists may, forexample, be identified following natural product screening or thescreening of chemical libraries. For natural product screening suitableenvironments include, but are not limited to, plants, bacteria and othermicroorganisms, river and sea beds, coral and arctic or antarcticregions. The present invention also contemplates antagonists directed toother components of the p75^(NTR)-mediated death signalling pathway.Such components to be targeted include but are not limited to Bcl-2 orrelated or homologous molecules. Preferably, for peptides, polypeptidesand proteins designed to induce cell death, the molecules arepalmitoylated.

[0110] Preferably, the peptide, polypeptide or protein comprises anamino acid sequence substantially as set forth in <400>8 or an aminoacid sequence having at least 60% identity thereto or a chemicalequivalent, derivative, homologue or analogue of said peptide,polypeptide or protein.

[0111] The term “isolated” means that the peptide, polypeptide orprotein of the present invention is provided in a form which is distinctfrom that which occurs in nature, preferably wherein one or morecontaminants have been removed. Accordingly, the isolated peptide,polypeptide or protein of the invention may be partially-purified orsubstantially pure, in which a substantial amount of the contaminantshave been removed or in sequencably pure or substantially homogeneousform.

[0112] The term “sequencably pure” means that the isolated peptide,polypeptide or protein is provided in a form which is sufficientlypurified to facilitate amino acid sequence determination usingprocedures known to those skilled in the art.

[0113] The term “substantially homogeneous” means that the isolatedpeptide, polypeptide or protein of the present invention is at leastabout 95% free of contaminants, more preferably at least about 99% freeof contaminants, including 100% purity.

[0114] The present invention extends to a range of derivatives andchemical analogues of the peptide, polypeptide or protein.

[0115] Furthermore, the amino acids of a homologous polypeptide may bereplaced by other amino acids having similar properties, for examplehydrophobicity, hydrophilicity, hydrophobic moment, charge orantigenicity, and so on.

[0116] “Analogues” encompass death signal containing peptides,polypeptides or proteins which are at least about 60% identical to thep75^(NTR) death signal sequence [<400>8], notwithstanding the occurrenceof any non-naturally occurring amino acid analogues therein. “Analogues”also encompass polypeptide mimotypes.

[0117] The term “derivative” in relation to a peptide, polypeptide orprotein shall be taken to refer hereinafter to mutants, parts orfragments derived from the functional p75^(NTR) molecule or death signalregion thereof or derivatives thereof which may or may not possess thedeath signal activity of the functional p75^(NTR). Derivatives includemodified peptides in which ligands are attached to one or more of theamino acid residues contained therein, such as carbohydrates, enzymes,proteins, polypeptides or reporter molecules such as radionuclides orfluorescent compounds. Glycosylated, fluorescent, acylated or alkylatedforms of the subject peptides are particularly contemplated by thepresent invention. Additionally, derivatives of the peptide, polypeptideor protein described herein comprise fragments or parts of an amino acidsequence disclosed herein are within the scope of the invention, as arehomopolymers or heteropolymers comprising two or more copies of thesubject polypeptides. Procedures for derivatizing peptides arewell-known in the art.

[0118] A homologue, analogue or derivative of <400>2 or <400>8 maycomprise an amino acid substitution or said <400>2 or 8 may encompassamino acid alterations in which an amino acid is replaced with adifferent naturally-occurring or a non-conventional amino acid residue.Such substitutions may be classified as “conservative”, in which case anamino acid residue contained in a phospholipase inhibitory protein isreplaced with another naturally-occurring amino acid of similarcharacter, for example Gly⇄Ala, Val⇄lle⇄Leu, Asp⇄Glu, Lys⇄Arg, Asn⇄Glnor Phe⇄Trp⇄Tyr.

[0119] Substitutions encompassed by the present invention may also be“non-conservative”, in which an amino acid residue which is present in aphospholipase inhibitory protein is substituted with an amino acidhaving different properties, such as a naturally-occurring amino acidfrom a different group (eg. substituted a charged or hydrophobic aminoacid with alanine), or alternatively, in which a naturally-occurringamino acid is substituted with a non-conventional amino acid.

[0120] Amino acid substitutions are typically of single residues, butmay be of multiple residues, either clustered or dispersed.

[0121] Naturally-occurring amino acids include those listed in Table 1.Non-conventional amino acids encompassed by the invention include, butare not limited to those listed in Table 2.

[0122] Amino acid deletions will usually be of the order of about 1-10amino acid residues, while insertions may be of any length. Deletionsand insertions may be made to the N-terminus, the C-terminus or beinternal deletions or insertions. Generally, insertions within the aminoacid sequence will be smaller than amino- or carboxyl-terminal fusionsand of the order of 1-4 amino acid residues. TABLE 1 Three-letterOne-letter Amino Acid Abbreviation Symbol Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln QGlutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V Any amino acid as above Xaa X

[0123] TABLE 2 Non-conventional amino acid Code α-aminobutyric acid Abuα-amino-α-methylbutyrate Mgabu aminocyclopropane- Cpro carboxylateaminoisobutyric acid Aib aminonorbornyl- Norb carboxylatecyclohexylalanine Chexa cyclopentylalanine Cpen D-alanine Dal D-arginineDarg D-aspartic acid Dasp D-cysteine Dcys D-glutamine Dgln D-glutamicacid Dglu D-histidine Dhis D-isoleucine Dile D-leucine Dleu D-lysineDlys D-methionine Dmet D-ornithine Dorn D-phenylalanine Dphe D-prolineDpro D-serine Dser D-threonine Dthr D-tryptophan Dtrp D-tyrosine DtyrD-valine Dval D-α-methylalanine Dmala D-α-methylarginine DmargD-α-methylasparagine Dmasn D-α-methylaspartate Dmasp D-α-methylcysteineDmcys D-α-methylglutamine Dmgln D-α-methylhistidine DmhisD-α-methylisoleucine Dmile D-α-methylleucine Dmleu D-α-methyllysineDmlys D-α-methylmethionine Dmmet D-α-methylornithine DmornD-α-methylphenylalanine Dmphe D-α-methylproline Dmpro D-α-methylserineDmser D-α-methylthreonine Dmthr D-α-methyltryptophan DmtrpD-α-methyltyrosine Dmty D-α-methylvaline Dmval D-N-methylalanine DnmalaD-N-methylarginine Dnmarg D-N-methylasparagine DnmasnD-N-methylaspartate Dnmasp D-N-methylcysteine Dnmcys D-N-methylglutamineDnmgln D-N-methylglutamate Dnmglu D-N-methylhistidine DnmhisD-N-methylisoleucine Dnmile D-N-methylleucine Dnmleu D-N-methyllysineDnmlys N-methylcyclohexylalanine Nmchexa D-N-methylornithine DnmornN-methylglycine Nala N-methylaminoisobutyrate NmaibN-(1-methylpropyl)glycine Nile N-(2-methylpropyl)glycine NleuD-N-methyltryptophan Dnmtrp D-N-methyltyrosine Dnmtyr D-N-methylvalineDnmval γ-aminobutyric acid Gabu L-t-butylglycine Tbug L-ethylglycine EtgL-homophenylalanine Hphe L-α-methylarginine Marg L-α-methylaspartateMasp L-α-methylcysteine Mcys L-α-methylglutamine MglnL-α-methylhistidine Mhis L-α-methylisoleucine Mile L-α-methylleucineMleu L-α-methylmethionine Mmet L-α-methylnorvaline MnvaL-α-methylphenylalanine Mphe L-α-methylserine Mser L-α-methyltryptophanMtrp L-α-methylvaline Mval N-(N-(2,2-diphenylethyl) Nnbhmcarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane L-N-methylalanine Nmala L-N-methylarginine NmargL-N-methylasparagine Nmasn L-N-methylaspartic acid NmaspL-N-methylcysteine Nmcys L-N-methylglutamine Nmgln L-N-methylglutamicacid Nmglu L-N-methylhistidine Nmhis L-N-methylisolleucine NmileL-N-methylleucine Nmleu L-N-methyllysine Nmlys L-N-methylmethionineNmmet L-N-methylnorleucine Nmnle L-N-methylnorvaline NmnvaL-N-methylornithine Nmorn L-N-methylphenylalanine NmpheL-N-methylproline Nmpro L-N-methylserine Nmser L-N-methylthreonine NmthrL-N-methyltryptophan Nmtrp L-N-methyltyrosine Nmtyr L-N-methylvalineNmval L-N-methylethylglycine Nmetg L-N-methyl-t-butylglycine NmtbugL-norleucine Nle L-norvaline Nva α-methyl-aminoisobutyrate Maibα-methyl-γ-aminobutyrate Mgabu α-methylcyclohexylalanine Mchexaα-methylcylcopentylalanine Mcpen α-methyl-α-napthylalanine Manapα-methylpenicillamine Mpen N-(4-aminobutyl)glycine NgluN-(2-aminoethyl)glycine Naeg N-(3-aminopropyl)glycine NornN-amino-α-methylbutyrate Nmaabu α-napthylalanine Anap N-benzylglycineNphe N-(2-carbamylethyl)glycine Ngln N-(carbamylmethyl)glycine NasnN-(2-carboxyethyl)glycine Nglu N-(carboxymethyl)glycine NaspN-cyclobutylglycine Ncbut N-cycloheptylglycine Nchep N-cyclohexylglycineNchex N-cyclodecylglycine Ncdec N-cylcododecylglycine NcdodN-cyclooctylglycine Ncoct N-cyclopropylglycine NcproN-cycloundecylglycine Ncund N-(2,2-diphenylethyl) Nbhm glycineN-(3,3-diphenylpropyl) Nbhe glycine N-(3-guanidinopropyl) Narg glycineN-(1-hydroxyethyl)glycine Nthr N-(hydroxyethyl))glycine NserN-(imidazolylethyl)) Nhis glycine N-(3-indolylyethyl) Nhtrp glycineN-methyl-γ-aminobutyrate Nmgabu D-N-methylmethionine DnmmetN-methylcyclopentytalanine Nmcpen D-N-methylphenylalanine DnmpheD-N-methylproline Dnmpro D-N-methylserine Dnmser D-N-methylthreonineDnmthr N-(1-methylethyl)glycine Nval N-methyla-napthylalanine NmanapN-methylpenicillamine Nmpen N-(p-hydroxyphenyl)glycine NhtyrN-(thiomethyl)glycine Ncys penicillamine Pen L-α-methylalanine MalaL-α-methylasparagine Masn L-α-methyl-t-butylglycine MtbugL-methylethylglycine Metg L-α-methylglutamate Mglu L-α-methylhomo Mhphephenylalanine N-(2-methylthioethyl) Nmet glycine L-α-methyllysine MlysL-α-methylnorleucine Mnle L-α-methylornithine Morn L-α-methylprolineMpro L-α-methylthreonine Mthr L-α-methyltyrosine Mtyr L-N-methylhomoNmhphe phenylalanine N-(N-(3,3-diphenylpropyl) Nnbhecarbamylmethyl)glycine

[0124] The present invention provides therefore, peptides, polypeptidesand proteins which inhibit p75^(NTR) death signalling and/or cleavage ofextracellular domain of p75^(NTR).

[0125] Accordingly, another aspect of the present invention contemplatesa method for inhibiting, reducing or otherwise antagonisingp75^(NTR)-mediated death signal in a neural cell, said method comprisingadministering a peptide, polypeptide or protein or analogues or mimeticsthereof which correspond to a non-membrane associated form of thep75^(NTR) death signal region or a derivative, functional equivalent orhomologue thereof.

[0126] Yet another aspect of the present invention is directed topeptide antagonists of the p75^(NTR) death signal or functionalanalogues or mimetics thereof.

[0127] The present invention provides for a method of treatment orprophylaxis of disease conditions associated with neural death or wherecell death is to be promoted such as in treating or preventing cancergrowth and/or development.

[0128] In one embodiment, it has been determined in accordance with thepresent invention that expression of a nucleic acid molecule encodingonly death signal and not adjacent, proximal or juxtaposed to amembrane-associating sequence results in antagonising of the deathsignal.

[0129] According to this embodiment, the present invention contemplatesa method for inhibiting, reducing or otherwise antagonising ap75^(NTR)-mediated death signal in a neural cell, said method comprisingintroducing a nucleic acid molecule capable of being expressed to anexpression product which corresponds to a non-membrane associated formof the p75^(NTR) death signal region or a derivative, functionalequivalent or homologue thereof.

[0130] In a related embodiment there is provided a method forinhibiting, reducing or otherwise antagonising a p75^(NTR)-mediateddeath signal in a neural cell, said method comprising contacting a cellcarrying a p75^(NTR) with a death signal-inhibiting effective amount ofa molecule capable of antagonising the death signal of p75^(NTR) or acomponent of the death signalling pathway.

[0131] This aspect of the present invention is useful for the treatmentof a range of neurodegenerative diseases such as cerebral palsy, traumainduced paralysis, vascular ischaemia associated with stroke, neuraltumours, motoneurone disease, Parkinson's disease, Huntington's disease,Alzheimer's disease, multiple sclerosis and peripheral neuropathiesassociated with diabetes, heavy metal or alcohol toxicity, renal failureand/or infectious diseases such as herpes, rubella, measles, chickenpox, HIV and/or HTLV-1. This aspect is also useful for treating neuronsor glia damaged by trauma or disease.

[0132] Alternatively, the method is aimed at targeting certain cellssuch as cancer cells wherein expression is required of a death signalfrom p75^(NTR) or a derivative, functional equivalent or homologuethereof adjacent, proximal or otherwise juxtaposed to amembrane-associating portion of p75^(NTR) or other membrane molecules oris in multimeric form. The nucleic acid molecule may requiremodification to ensure appropriate targeting to the cell or the nucleicacid molecule may be injected directly into cancerous tissue.

[0133] Another aspect of the present invention provides a biologicalcomposition comprising a genetic molecule capable of being expressedinto a p75^(NTR) death signal antagonist or a p75^(NTR) death signal.The biological composition further comprises one or morepharmaceutically acceptable carriers andlor diluents. The nucleic acidmolecules according to this aspect of the present invention may be nakednucleic acid molecules or contained or associated with a viral vector orother suitable delivery mechanism.

[0134] Another aspect of the present invention is directed to abiological composition comprising a molecule capable of antagonisingp75^(NTR)-mediated death signalling of a cell.

[0135] Suitable molecules according to this aspect of the presentinvention are as contemplated above and include a peptide, polypeptideor protein comprising a soluble form of the p75^(NTR) death signallingregion or an antagonist of a component of the p75^(NTR) death signallingpathway.

[0136] The present invention is also useful as a culture agent such aspreventing or reducing the death of cells in vitro. The presentinvention is particularly useful in vitro when used in combination withLIF. Even more particularly, the present invention is useful forculturing recombinant cell lines.

[0137] The present invention also provides for the use of the deathsignal of p75^(NTR) in the manufacture of a medicament for the treatmentof neurodegenerative diseases in animals. Preferred animals includehumans, primates, livestock animals, laboratory test animals, companionanimals and captive wild animals.

[0138] The present invention is further described by the followingnon-limiting Examples.

EXAMPLE 1

[0139] The aim of this example was to determine the protein domains onp75^(NTR) responsible for death signalling.

[0140] In order to investigate how p75^(NTR) signals neural death, theinventors devised a robust in vitro assay for p75^(NTR) induced death.Plasmid expression constructs were microinjected into individual neuronsin the presence of the growth factor LIF, and the survival of theneurons expressing the different plasmids was determined. A series ofplasmid constructs which encode incomplete p75^(NTR) proteins were made(see FIG. 1) and the ability of each protein to signal death when overexpressed was assessed.

[0141] The p75^(NTR) protein is a transmembrane protein comprised of alarge extracellular domain with four cysteine rich motifs responsiblefor interacting with soluble growth factors, and a short cytoplasmic,intracellular tail. The cytoplasmic domain does not contain a kinasedomain but contains a domain with significant homology to a motif knownas a “death domain” [<400>9, <400>10], found in apoptosis-inducingTumour Necrosis Factor Receptors (TNFR) and TNFR-associatingdeath-effector proteins [9].

[0142] Using expression plasmids of p75^(NTR) proteins deleted foreither the entire cytoplasmic domain (p75nc) or a significant portion ofthe cytoplasmic domain including the entire death domain (p75tm), theinventors found that the cytoplasmic domain is responsible for deathsignalling. Surprisingly, the intracellular 35 amino acid domainjuxtaposed to the membrane, and not the death domain, is responsible fordeath signalling (FIG. 2). This region of the p75^(NTR) protein shows nohomology to other death inducing proteins or to known functional motifs.

[0143] To further investigate the domain required for death signallingthe inventors made constructs expressing p75^(NTR) proteins deleted forthe extracellular domain or the extracellular and transmembrane domains.Proteins without extracellular domains retain the signal peptide whichis responsible for correctly transporting the protein into the cellmembrane. Proteins without transmembrane domains are expressed free inthe cytoplasm of the cell and are epitope tagged with a FLAG motif fordetection.

[0144] The inventors found that the extracellular domain of p75^(NTR)had a significant inhibitory effect of the ability of the cytoplasmicdomain to signal cell death. Furthermore, the membrane linked 35 aminoacid cytoplasmic domain (c35) was a potent stimulant of neural deathwith over 90% of cells injected with the plasmid dead after 16 hours(FIG. 3). However, if the cytoplasmic 35 amino acid domain is notassociated with the membrane, the ability of the domain to induce deathis removed (FIG. 4).

[0145] These results indicate that the domain responsible for deathinduction is within the first 35 amino acids of the cytoplasmic tail butthat the transmembrane domain, or at least association with themembrane, is required for death-signal activation. This may be relatedto the ability of the transmembrane protein to more efficiently formdeath-signal inducing multimers, or that the position of the p75^(NTR)protein in relation to other membrane-bound accessory molecules might beimportant in initiating death signalling.

[0146] The inventors hypothesised that the free cytoplasmic expressed 35amino acid domain might be able to interfere with death signalling fromfull length p75^(NTR) proteins by a dominant-negative mechanism, andattempted to inhibit the death by co-expressing the proteins. Given theresults presented below regarding the ability of overexpression of Bcl-2to enhance p75^(NTR) killing, this paradigm was used to test the abilityof the c35 protein to inhibit death signalling. The inventors found thatindeed the expression of the c35 protein was able to inhibit thiskilling (FIG. 5). This further indicates that p75^(NTR) signals killingvia interaction of an accessory molecule to a motif within the first 35amino acids of the cytoplasmic domain.

EXAMPLE 2

[0147] The aim of this example is to determine the minimum number ofamino acid residues on c35 require to mediate death signalling.

[0148] A series of deletion and truncation mutants in the c35 region areproduced and tested for the ability to induce death signalling.

[0149] The deletion mutants from the membrane distal end are as follows:KRWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSG; KRWNSCKQNKQGANSRPVNQTPPPEGEKLHSDS;KRWNSCKQNKQGANSRPVNQTPPPEGEKLHSD; KRWNSCKQNKQGANSRPVNQTPPPEGEKLHS;KRWNSCKQNKQGANSRPVNQTPPPEGEKLH; KRWNSCKQNKQGANSRPVNQTPPPEGEKL;KRWNSCKQNKQGANSRPVNQTPPPEGEK; KRWNSCKQNKQGANSRPVNQTPPPEGE;KRWNSCKQNKQGANSRPVNQTPPPEG; KRWNSCKQNKQGANSRPVNQTPPPE;KRWNSCKQNKQGANSRPVNQTPPP; KRWNSCKQNKQGANSRPVNQTPP;KRWNSCKQNKQGANSRPVNQTP; KRWNSCKQNKQGANSRPVNQT; KRWNSCKQNKQGANSRPVNQ;KRWNSCKQNKQGANSRPVN; KRWNSCKQNKQGANSRPV; KRWNSCKQNKQGANSRP;KRWNSCKQNKQGANSR; KRWNSCKQNKQGANS; KRWNSCKQNKQGAN; KRWNSCKQNKQGA;KRWNSCKQNKQG; KRWNSCKQNKQ; KRWNSCKQNK; KRWNSCKQN; KRWNSCKQ; KRWNSCK;KRWNSC; KRWNS; KRWN; KRW; KR; and K.

[0150] The deletion mutants from the membrane proximal end are asfollows: RWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGI;WNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGI; NSCKQNKQGANSRPVNQTPPPEGEKLHSDSGI;SCKQNKQGANSRPVNQTPPPEGEKLHSDSGI; CKQNKQGANSRPVNQTPPPEGEKLHSDSGI;KQNKQGANSRPVNQTPPPEGEKLHSDSGI; QNKQGANSRPVNQTPPPEGEKLHSDSGI;NKQGANSRPVNQTPPPEGEKLHSDSGI; KQGANSRPVNQTPPPEGEKLHSDSGI;QGANSRPVNQTPPPEGEKLHSDSGI; GANSRPVNQTPPPEGEKLHSDSGI;ANSRPVNQTPPPEGEKLHSDSGI; NSRPVNQTPPPEGEKLHSDSGI; SRPVNQTPPPEGEKLHSDSGI;RPVNQTPPPEGEKLHSDSGI; PVNQTPPPEGEKLHSDSGI; VNQTPPPEGEKLHSDSGI;NQTPPPEGEKLHSDSGI; QTPPPEGEKLHSDSGI; TPPPEGEKLHSDSGI; PPPEGEKLHSDSGI;PPEGEKLHSDSGI; PEGEKLHSDSGI; EGEKLHSDSGI; GEKLHSDSGI; EKLHSDSGI;KLHSDSGI; LHSDSGI; HSDSGI; SDSGI; DSGI; SGI; GI; and I.

EXAMPLE 3 Role of Bcl-2 in Promoting p75^(NTR) Mediated Death Signalling

[0151] As the inventors had shown that the death of dorsal root ganglia(DRG) sensory neurons in vitro and in vivo, was, at least in part,mediated by p75^(NTR), p75^(NTR) was over-expressed in these cells bymicroinjecting rat p75^(NTR) cDNA expressing plasmid into the nucleus ofmouse sensory neurons. These were cultured in the presence of the LIF toprevent neural death not linked to p75^(NTR) mechanisms. It was foundthat the expression of the rat p75^(NTR) could be detected by surfaceimmunofluorescence within 24 hours of injection. The injected neuronswere observed over a 48 hour period and the viability was assessed. Itwas found that within the first 16 hours, a significantly higher numberof p75^(NTR) plasmid injected neurons had died compared to neuronsinjected with control plasmids β-galactosidase, or a truncated p75^(NTR)protein lacking the entire cytoplasmic domain (p75^(NTR)nc). It wasfound that p75^(NTR)-mediated neural death occurred later in theexperiment similar to Fas/TNF-induced rapid cell death. Since bothfull-length p75^(NTR) and p75^(NTR)nc protein showed a similar level ofexpression after injection, this indicates that the cytoplasmic domainof p75^(NTR) is required for death signalling. This was expected sincethe cytoplasmic tail contains a sequence with homology to the Fas/TNFR“death domain” [9].

[0152] The inventors next examined whether deletion of the “deathdomain” also abolished the ability of p₇₅ ^(NTR) to kill. It was foundthat the neural death observed after expression of p75^(NTR) with atruncated cytoplasmic tail (p75^(NTR)tr) was equivalent to thefull-length p75^(NTR) protein. This demonstrated that the “death domain”was not required for p75^(NTR) killing and, since the p75^(NTR) deathdomain has recently been shown to have a different tertiary structure toTNFR family death domain and does not self-associate in vitro, itsuggests that the p75^(NTR) “death domain” may not normally function toinduce death. Together, these results predict that an alternativepathway involving proteins other than “death domain” adapter proteins,such as TRADD and FADD, is responsible for p75^(NTR)-mediated killing.

[0153] The Bcl-2 family of proteins is involved in mediating apoptoticsignalling pathways, and can homodimerise or heterodimerise with otherfamily members. Bcl-2 and Bcl-xL are well characterised inhibitors ofstress-induced apoptosis, JNK activation and neural death due togrowth-factor limitation. However, both are poor inhibitors of Fas andTNFR mediated opoptosis. As it had been shown previously that highlevels of Bcl-2 or Bcl-xL blocked neural cell death in a variety ofmodels, the inventors examined whether over-expression of these proteinscould block the death induced by p75^(NTR).

[0154] The inventors found that over-expression of Bcl-xL protectedneurons against p75^(NTR)-induced death, supporting the hypothesis thatp75^(NTR) signals through an alternative pathway to TNFR-inducedapoptosis. In contrast, while Bcl-2 over-expression alone had no effecton cell survival in the presence of LIF, Bcl-2 in combination withp75^(NTR) over-expression, surprisingly, induced a significant increasein neural death above that seen with p75^(NTR) over-expression alone.Bcl-2 in combination with p75^(NTR)nc did not cause significant celldeath and furthermore, the cell death observed with p75^(NTR) and Bcl-2over-expression was totally ablated if the cells were cultured in NGF.Bcl-2 was able to protect against neural death induced by NGFwithdrawal, but not withdrawal of LIF. Thus, at the same expressionlevels in the same neural population, Bcl-2 was able to prevent orenhance neural cell death depending on the nature of the death signal.

[0155] These results are surprising since Bcl-2 has previously beenshown to have similar actions to Bcl-xL in almost all cell-deathsystems.

[0156] To determine whether the paradoxical effect of Bcl-2 onp75^(NTR)-induced killing was related to its known anti-apoptoticactivity, Bcl-2 proteins with inactivating point mutation, G145E, in the“Bcl-2 Homology” BH1 domain and W188A in the BH2 domain were utilised.Like wildtype Bcl-2, expression of either Bcl-2 mutant alone did noteffect neural survival. In combination with p75^(NTR) expression, theenhanced killing effect seen with Bcl-2 co-expression was abrogated bythe G145E mutation, even though the proteins were expressed tocomparable levels. Thus, an intact BH1 homology region is required forthe death promoting activity of Bcl-2.

[0157] Mutation of the equivalent G138 residue in Bcl-xL results in aconformational change between α-helices 4 and 5, disrupting access tothe hydrophobic cleft formed by BH1, BH2 and BH3 domains. Therefore, themolecular mechanism by which Bcl-2 participates in the p75^(NTR) killingpathway may be dependent on interactions either directly with the BHdomains or with the hydrophobic cleft, as indicated with experimentsusing the W188A mutation. Co-expression of p75^(NTR) with the Bcl-2W188A protein not only abrogated the increased p75^(NTR) killing but,more importantly, protected neurons from any p75^(NTR)-induced death,reminiscent of that seen with Bcl-xL. These experiments suggest that theconformation of the Bcl-2 protein is integral to the opposing functionsobserved herein.

[0158] The inventors had observed that DRG neurons isolated from newbornmice depleted for p75^(NTR) were less susceptible to NGF withdrawal, asis the case with sympathetic neurons, when compared to neurons fromwildtype mice. This is indicative of absent or delayed naturallyoccurring cell death observed in these mice. The inventors attempted toinduce cell death in p75^(NTR) “knock out” DRG neurons by re-introducingp75^(NTR) expression. Surprisingly, apoptosis was not induced byre-expression into “knock out” DRG neurons, the inventors found thatneural death was significantly increased under these conditions. Thisimplicated an absolute requirement for Bcl-2 in mediating p75^(NTR)killing.

[0159] The inventors tested, therefore, whether high endogenous Bcl-2levels might be necessary for successful p75^(NTR)-mediated killing innormal neurons by assaying p75^(NTR) killing in Bcl-2 depleted cells.Endogenous Bcl-2 was down regulated by antisense as previouslydescribed. When the Bcl-2 antisense plasmid was injected at the sametime as p75^(NTR) plasmids no diminishment in the death signal was seen.If, however, the Bcl-2 antisense was microinjected first (to give timeto reduced Bcl-2 production and deplete endogenous Bcl-2; and then a daylater the p75^(NTR) or p75^(NTR)nc constructs were microinjected, therewas no difference in survival between p75^(NTR) and p75^(NTR)ncexpressing neurons, strongly suggesting that endogenous Bcl-2 isrequired for p75^(NTR) killing effects. To confirm this observation, theinventors isolated neurons from newborn Bcl-2 “knock out” mice (anheterozygous line of mice containing a disrupted Bcl-2 gene) and theirwild-type litter mates and compared the effect of p75^(NTR)over-expression with control plasmid p75^(NTR). It was found that theneurons isolated from Bcl-2 deficient mice were significantly protectedfrom p75^(NTR) killing, showing a 56.9% (n=3) reduction in deathcompared wildtype neurons, supporting the hypothesis that endogenousBcl-2 is required for p75^(NTR) killing.

[0160] Bcl-2 has previously been observed to increase cell death whenhighly expressed both in vitro and in vivo when expressed at high levelsas a transgene, causing increased apoptosis in the brain under a neuronspecific promoter, or in photoreceptor cells when expressed specificallyunder a rhodopsin promoter. Thus, it is possible that the high level ofBcl-2 is able to “prime” the death pathway such that an apoptoticstimulus via p75^(NTR) results in rapid cell death.

[0161] Bcl-2 and Bcl-xL when cleaved by caspases have also been shown tobe capable of promoting apoptosis in vitro, with cells expressingnon-cleavable mutant Bcl-2 and Bcl-xL proteins showing increasedviability compared to cells expressing wildtype proteins. Cleavage ofBcl-2 is possible in this system, however, the Bcl-2 mutations whichresults in loss of death promoting activity, would not prevent cleavageof Bcl-2, indicating that cleavage of Bcl-2 would only be part of themechanism by which Bcl-2 promotes killing. In addition, if cleavage wasthe dominant mechanism, Bcl-xL might be expected to act as a deathsignalling protein in this system.

[0162] To investigate whether the p75^(NTR)-Bcl-2 death-signallingcascade was dependent on caspase activation, inhibitors of caspases wereemployed. In the presence of z-VAD, a nonspecific caspase peptideinhibitor, or after co-expression of modified crmA plasmids, designed toinhibit Group II caspases such as caspases 2 and 3, p75^(NTR)-mediateddeath was significantly reduced. Similarly, the modified crmA was ableto block the killing induced by co-expression of p75^(NTR) and Bcl-2.This indicates that p75^(NTR) induced apoptosis is a caspase dependentpathway and that the mechanism by which Bcl-2 assists killing is throughthe same pathway.

EXAMPLE 4

[0163] Antagonism of p75^(NTR) Mediated Death Signalling

[0164]FIG. 6 shows that soluble c35 (35 mer) [<400>7 and <400>8]protects cells from death signalling in a dose-dependent manner againstmembrane bound c35. Furthermore, the 35 mer when expressed from agenetic construct, protected Schwann cells against NGF-induced death.c35 when expressed in soluble form can also protect cells againstmembrane bound c35. The inventors show in FIG. 7 that soluble c35 canalso protect against membrane-linked, expressed c35. A truncated form ofc35, a 29 mer [<400>11 and <400>12], also protected againstmembrane-bound c35, when in soluble form.

[0165]FIG. 8 shows that the 29 mer with a palmitoyl group at themembrane (amino) end resulted in cell death. The palmitoylation linksthe peptide to the plasma membrane. This membrane-linked 29 mer leads tocell death whereas its soluble form protects cells againstp75^(NTR)-mediated death signalling.

EXAMPLE 5 Effects of Palmitoylation

[0166]FIG. 9 shows the effects of palmitoylated 29 mer (fused topenetratin) on mediating cell death. Cells were washed with peptide (2μM) for approximately 110 minutes and the cells were then washed.Controls included penetratin-fused 29 mer, palmitoylatedpenetratin-fused 29 mer palmitoylated penetratin-fused gp130 andpenetratin alone. Palmitoylated, penetratin fused 29 mer mediatedsignificant cell death.

EXAMPLE 6 Passage Across Blood Brain Barrier

[0167] The ability for peptides to cross the blood brain barrier istested using fluorescence-linked peptides injected intraperitonealy intomice. The peptides may be fused to penetratin or fused or associatedwith the TAT protein from HIV (18).

EXAMPLE 17 Animal Models

[0168] Peptides are delivered with penetratin or TAT (18) to variousanimal models for neurodegenerative diseases. The animal models usedinclude: Animal Model Disease Axotomy of newborn rat sensory Peripheralneuropathies neurons Axotomy of newborn rat motor neurons Motor neurondisease (SOD1 mice: B6SJL-TgN [SOD1- G93A] 1 Gurd1) Ischemia of adultrats Stroke Experimental allergic encephamylitis Multiple sclerosis andoptic nerve axotomy [19]

[0169] Those skilled in the art will appreciate that the inventiondescribed herein is susceptible to variations and modifications otherthan those specifically described. It is to be understood that theinvention includes all such variations and modifications. The inventionalso includes all of the steps, features, compositions and compoundsreferred to or indicated in this specification, individually orcollectively, and any and all combinations of any two or more of saidsteps or features.

1. An isolated nucleic acid molecule comprising a sequence ofnucleotides or complementary sequence of nucleotides which encodes anamino acid sequence which is capable of signalling, inducing orotherwise facilitating the death of a cell in which said amino acidsequence is adjacent, proximal or otherwise juxtaposed to the membraneof said cell or when said amino acid is in multimeric form.
 2. Anisolated nucleic acid molecule according to claim 1 wherein the aminoacid sequence comprises a membrane-associating portion and/ormultimer-forming portion and a portion which corresponds to all or partof the cytoplasmic region of p75^(NTR) or a functional equivalent,derivative or homologue thereof.
 3. An isolated nucleic acid moleculeaccording to claim 2 wherein the membrane-associated portion is derivedfrom p75^(NTR) or a functional equivalent, derivative or homologuethereof.
 4. An isolated nucleic acid molecule according to claim 2wherein the membrane-associating portion is from a receptor or otherligand-binding molecule.
 5. An isolated nucleic acid molecule accordingto claim 4 wherein the receptor or other ligand-binding molecule is acytokine receptor, immunoglobulin or T-cell receptor.
 6. A homologue,analogue or derivative of the nucleic acid molecule of any one of claims1 to
 5. 7. An isolated nucleic acid molecule comprising a sequence ofnucleotides which encodes an amino acid sequence which inhibits orreduces p75^(NTR)-mediated cell death wherein said amino acid sequenceis a soluble form of the p75^(NTR) receptor corresponding to anintracellular region adjacent, proximal or otherwise juxtaposed to themembrane of said cell.
 8. An isolated nucleic acid molecule according toclaim 7 wherein the p75^(NTR) is of human, primate or murine origin. 9.An isolated nucleic acid molecule according to any one of the proceedingclaims comprising a nucleotide sequence substantially capable ofhybridizing to <400>1 or its complementary form under low stringencyconditions.
 10. An isolated nucleic acid molecule according to claim 9comprising a nucleotide sequence substantially as set forth in <400>7 ora nucleotide sequence capable of hybridizing to <400>7 or itscomplementary form under low stringency conditions or a nucleotidesequence having at least 60% identity to <400>7.
 11. An isolated nucleicacid molecule according to claim 9 comprising a nucleotide sequenceencoding an amino acid sequence set forth in <400>8 or an amino acidsequence having at least 60% identity thereto.
 12. A nucleic acidmolecule comprising the nucleotide sequence: {n₁ - - -n_(x)}_(b)a{n′_(l) - - - n′_(y)}_(c)a{n″_(l) - - - n″_(z)}_(d) wherein{n₁ - - - n_(x)} is a sequence of x nucleotides encoding anextracellular portion of a receptor or ligand-binding molecule;{n′_(l) - - - n′_(y)} is a sequence of y nucleotides encoding atransmembrane peptide, polypeptide or protein or a molecule capable ofinducing multimerisation; {n″_(l) - - - n″_(z)} is a sequence of znucleotides comprising a nucleotide sequence substantially as set forthin <400>7 or a nucleotide sequence encoding an amino acid sequencesubstantially as set forth in <400>8 or a nucleotide sequence capable ofhybridising to <400>7 or a complementary form thereof under lowstringency conditions such as at 42° C. or a nucleotide sequence havingat least 60% identity to <400>7; b, c and d may be the same ordifference and each is 0, 1 or >1; x, y and z may be the same ordifferent and each is 0, 1 or >1; a is a nucleotide bond; wherein when cis 1 or >1 and d is 1 or >1 and wherein when the molecule is expressedin a neural cell, the expression product signals, induces or otherwisefacilitates cell death.
 13. A nucleic acid molecule according to claim12 wherein {n₁ - - - n_(x)} comprises the nucleotide sequencesubstantially as set forth in <400>3 or is a nucleotide sequence havingat least about 60% identity thereto or is capable of hybridising theretounder low stringency conditions at 42° C.
 14. A nucleic acid moleculeaccording to claim 12 wherein {n′_(l) - - - n′_(y)} comprises thenucleotide sequence substantially as set forth in <400>5 or is anucleotide sequence having at least about 60% identity thereto or iscapable of hybridising thereto under low stringency conditions at 42° C.15. A genetic construct comprising an isolated nucleic acid moleculewhich comprises a sequence of nucleotides which corresponds or iscomplementary to a death signal region from p75^(NTR) or a homologue,analogue or derivative thereof.
 16. A genetic construct according toclaim 15 wherein the coding region of the death signal from p75^(NTR) isplaced in operable connection with a promoter sequence such that a geneproduct is capable of being expressed under the control of said promotersequence.
 17. A genetic construct according to claim 15 or 16 whereinsaid genetic construct further comprises a terminator sequence.
 18. Amethod for inhibiting, reducing or otherwise antagonisingp75^(NTR)-mediated death signal in a neural cell, said method comprisingadministering a peptide, polypeptide or protein or analogues or mimeticsthereof which correspond to a non-membrane associated form of thep75^(NTR) death signal region or a derivative, functional equivalent orhomologue thereof.
 19. A peptide antagonist of the p75^(NTR) deathsignal or functional analogues or mimetics thereof.
 20. A recombinantpeptide, polypeptide or protein produced by expressing the isolatednucleic acid molecule according to any one of claims 1 to 14 in asuitable host cell or a derivative, homologue or analogue of saidpeptide, polypeptide or protein.
 21. An isolated peptide, polypeptide orprotein according to claim 20 comprising the cytoplasmic region ofp75^(NTR) which signals, induces or otherwise facilitates cell deathwhen said peptide, polypeptide or protein is adjacent, proximal orotherwise juxtaposed to a membrane-associating region such as fromp75^(NTR) or other membrane molecule and/or is in multimeric form or aderivative, homologue, chemical equivalent or analogue of said peptide,polypeptide or protein.
 22. A peptide, polypeptide or protein accordingto claim 21 comprising an amino acid sequence substantially as set forthin <400>8 or an amino acid sequence having at least 60% identity theretoor a chemical equivalent, derivative, homologue or analogue of saidpeptide, polypeptide or protein.
 23. A method for inhibiting, reducingor otherwise antagonising a p75^(NTR)-mediated death signal in a neuralcell, said method comprising introducing a nucleic acid molecule capableof being expressed to an expression product which corresponds to anon-membrane associated form of the p75^(NTR) death signal region or aderivative, functional equivalent or homologue thereof.
 24. A method forinhibiting, reducing or otherwise antagonising a p75^(NTR)-mediateddeath signal in a neural cell, said method comprising contacting a cellcarrying a p75^(NTR) with a death signal-inhibiting effective amount ofa molecule capable of antagonising the death signal of p75^(NTR) or acomponent of the death signalling pathway.
 25. A method according toclaim 23 or 24 for the treatment of a range of neurodegenerativediseases such as cerebral palsy, trauma induced paralysis, vascularischaemia associated with stroke, neural tumours, motoneurone disease,Parkinson's disease, Huntington's disease, Alzheimer's disease, multiplesclerosis and peripheral neuropathies associated with diabetes, heavymetal or alcohol toxicity, renal failure and/or infectious diseases suchas herpes, rubella, measles, chicken pox, HIV and/or HTLV-1.
 26. Amethod according to claim 23 or 24 for treating neurons and neural cellsdamaged by trauma or disease.
 27. A biological composition comprising agenetic molecule capable of being expressed into a p75^(NTR) deathsignal antagonist or a p75^(NTR) death signal said composition furthercomprising one or more pharmaceutically acceptable carriers and/ordiluents.
 28. A method for modulating p75^(NTR)-mediated death signal ina neural cell, said method comprising administering an agent whichantagonises or agonises cleavage of the extracellular domain ofp75^(NTR).
 29. Use of an antagonist of the death signal of p75^(NTR) inthe manufacture of a medicament for the treatment of neurodegenerativediseases in animals.
 30. Use according to claim 29 wherein the animal isa human, primate, livestock animal, laboratory test animal, companionanimal and/or captive wild animal.